Editorial For reprint orders, please contact [email protected]
Polysialic acid: overcoming the hurdles of drug delivery “In the coming years, we should expect to see a surge in carrier systems based upon inherently biodegradable, nonimmunogenic, natural materials, such as polysialic acid...” Keywords: drug delivery n nanocarriers n PEG n polysialic acid
“I never really worried about those hurdles. They were just standing there and I was always zooming past them just to get back on the ground again.” – Rodney Milburn (1972 110 m hurdle Olympic champion) In the past two decades, PEG has been lauded as the gold standard material in generating carrier systems that can extend the circularly stability of associated therapeutics and facilitate passive accumulation within diseased tissue [1]. Most investigators take for granted the biocompatibility and aqueous solubility of this synthetic polymer. However, if we step back and take a closer look at PEG, the plausibility that this is the ultimate solution to overcome the hurdles associated with drug delivery is called into question. Although widely regarded as nonimmunogenic, an increasing number of reports suggest that PEG is capable of inducing an immune response in both animal models and human patients, particularly when administered repeatedly. For example, Semple et al. observed a generation of PEG-reactive plasma IgM after dosing mice with PEG-liposomes [2]. Likewise, a recent study by Hamad and coworkers reported concentration- and molecular-weight-dependent activation of the complement system by PEG [3]. When the potential for immunogenicity is combined with a lack of degradability, PEG is a particularly poor choice for drug delivery in the treatment of immune system disorders and/or diseases that require long term treatment, such as rheumatoid arthritis. Of note, low-molecularweight PEG can be metabolized by alcohol and aldehyde dehydrogenase; however, the resultant dicarboxylic acid and hydroxyl-carboxylic acid metabolites are toxic and can lead to acidosis, as indicated in human and animal studies [4]. Natural polymers, with inherent biodegradability and nonimmunogenicity, have been sought after for use in drug delivery as a means
to evade the problems associated with synthetic polymer use, namely, the lack of degradability and the potential of inducing an immune response. Although largely unheralded by other investigators until recent years, in 1993, Gregoriadis posited that polysialic acid (PSA), a highly hydrophilic polysaccharide composed primarily of a-2,8-linked 5-N-glycolyneuraminic acid, could be used as a suitable PEG alternative. Similar to the fixed aqueous layer associated with PEG-modified compounds, PSA is thought to be surrounded by a protective envelope of water, referred to as the ‘watery cloud’, which prevents interactions with cells and proteins [5]. Thus, PSA possesses the requisite properties to impart a stealth nature on associated molecules. In mammalian tissue, neural cell adhesion molecules are post-translationally modified by PSA, and the negatively charged polymer chains are thought to diminish cell–cell interactions, thereby promoting neural plasticity. PSA is assumed to act in a nearly identical fashion to promote tumor growth and cancer metastasis [6]. In bacteria, PSA, with repeating units identical to those found in PSA produced by mammalian cells, is used to mask the invading organism from the host immune system [7,8]. Thus, taken together, PSA acts in nature to reduce undesirable cellular interactions and prevent recognition by the reticuloendothelial system, the exact traits necessary for an effective drug-delivery system. Initia l proof-of-concept studies by Gregoriadis et al. using PSA conjugated to a fluorescent tag verified that the half-life of PSA approached 40 h following intravenous administration to mice [5]. Extending this research to the delivery of therapeutic proteins, the half-lives of asparaginase, insulin, GCSF and IFN-a2b in animal models were significantly increased via polysialylation [9,10]. Moreover, asparaginase did not elicit the immune response that was observed following administration of free asparaginase
10.4155/TDE.13.153 © 2014 Future Science Ltd
Ther. Deliv. (2014) 5(3), 235–237
Rebecca A Bader Author for correspondence: Department of Biomedical & Chemical Engineering, Syracuse Biomaterials Institute, Syracuse University, Syracuse, NY, 13244 USA Tel.: +1 315 857 4622 Fax: +1 315 443 9175 [email protected]
Patricia R Wardwell Department of Biomedical & Chemical Engineering, Syracuse Biomaterials Institute, Syracuse University, Syracuse, NY, 13244, USA
ISSN 2041-5990
235
Editorial | Bader & Wardwell [11]. Based upon polysialylation technology, Gre-
goriadis founded and now serves as Chief Scientific Offer for Xenetic Biosciences (London, UK). Through partnering with biotechnology and pharmaceutical companies, clinical trials are currently being conducted with PSA conjugates of erythropoietin and insulin for the treatment of anemia and diabetes, respectively, while PSA conjugates of factor VIII (hemophilia) and deoxyribonuclease (cystic fibrosis) are in the preclinical stage of development [12]. Although the latter technology relies upon conjugation via standard synthetic methodology, an emerging approach to the synthesis of PSA-modified proteins utilizes the production of recombinant glycoproteins by engineering the protein glycosylation pathways in Escherichia coli [13]. In addition to exploring new methods of synthesizing protein–PSA conjugates, investigators are also looking into how the efficacy of small-molecule therapeutics can be improved via conjugation to PSA [14].
“...polysialic acid acts in nature to reduce undesirable cellular interactions and prevent recognition by the reticuloendothelial system, the exact traits necessary for an effective drug-delivery system.” PSA has also been used in a manner similar to PEG in coating colloidal-carrier systems, which have higher loading capacity and minimal risk of altering drug activity relative to conjugates. Gregoriadis et al. has explored the use of polysialic acid coatings to extend the circulation time of liposomes polysialylation [9,10]. However, some concerns remain pertaining to the utility of PSA coatings. For instance, a change in the conformation of PSA, as can occur following binding to the surface of a carrier system, can lead to an undesirable immunogenic response as a result of a change in the tertiary structure [15]. Notably, coating with sialic acid monomers has largely been unsuccessful in improving the targeted delivery of therapeutics with colloidal carrier systems using animal models, suggesting that PSA, rather than the monomeric form, should be used to extend physiological stability [16,17]. Instead of application of a PSA coating, the authors have focused upon the development of colloidal-carrier systems that utilize PSA as the basis. Micelles have been prepared via self-assembly 236
Ther. Deliv. (2014) 5(3)
following grafting of hydrophobic molecules onto the PSA backbone. Although initial studies used long-chain hydrocarbons, particularly decylamine [18], current research is focused upon use of polycaprolactone (PCL) as the hydrophobic molecule. As expected, based upon Gregoriadis’s ‘watery cloud’ theory, the PSA shell yielded a fixed aqueous layer thickness similar to that obtained for PEG-coated liposomes [19]. The noncytotoxic PSA–PCL system is being explored for use in drug delivery to improve treatment strategies for rheumatoid arthritis and cardiovascular disease. Furthermore, this system can be readily modified by a variety of targeting moieties to facilitate active targeting of diseased tissue. For example, hyaluronic acid has been conjugated to the PSA–PCL surface for enhanced targeting of the hyaluronic acid CD44 receptor, which is overexpressed by certain cell types in cancer, rheumatoid arthritis and cardiovascular disease. PSA has also been used to prepare nanoparticles via complexation with N-trimethylchitosan (TMC). In contrast to existing polysaccharide-based nanoparticles, for the most part, PSA–TMC nanoparticles possess a smaller size, that is more appropriate for drug delivery and a lower polydispersity [20]. The PSA–TMC nanoparticles are being explored for use in the delivery of conventional and new biologic therapeutics for the treatment of rheumatoid arthritis and cystic fibrosis [Wardwell PR, Bader RA, Unpublished Data]. In vivo experiments are currently underway to verify that the PSA-based carrier systems are non-immunogenic and can be used to extend the half-lives and enhance the efficacy of associated therapeutics similar to observations for PSA-conjugates. For a number of years, researchers have seemingly been attempting to overcome the hurdles to drug delivery through the use of a readily available, synthetic polymer, PEG. However, the easiest way to clear a hurdle is not always the best way, particularly for achieving long-term success. Researchers have begun to search nature for the solutions to problems that continue to plague biomaterials, and materials used for drug delivery are not exempt from this search. Although Gregoriadis first suggested PSA as the most rational choice for improved drug delivery 20 years ago, only recently have other investigators truly begun to take notice. In the coming years, we should expect to see a surge in carrier systems based upon inherently biodegradable, nonimmunogenic, natural materials, such as PSA, that can move through the body undetected to better facilitate targeted delivery. future science group
Polysialic acid: overcoming the hurdles of drug delivery Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes
| Editorial
employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
References 1
2
3
4
5
6
7
Fishburn CS. The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics. J. Pharm. Sci. 97(10), 4167–4183 (2008). Semple SC, Harasym TO, Clow KA, Ansell SM, Klimuk SK, Hope MJ. Immunogenicity and rapid blood clearance of liposomes containing polyethylene glycol-lipid conjugates and nucleic acids. J. Pharmacol. Exp. Ther. 312(3), 1020–1026 (2005). Hamad I, Hunter AC, Szebeni J, Moghimi SM. Poly(ethylene glycol)s generate complement activation products in human serum through increased alternative pathway turnover and a MASP-2-dependent process. Mol. Immunol. 46(2), 225–232 (2008). Webster R, Didier E, Harris P et al. PEGylated proteins: evaluation of their safety in the absense of definitive metabolism studies. Drug Metab. Dispos. 35(1), 9–16, (2007). Gregoriadis G, Mccormack B, Wang Z, Lifely R. Polysialic acids: potential in drug delivery. FEBS Lett. 315(3), 271–276 (1993). Dube DH, Bertozzi CR. Glycans in cancer and inflammation. Potential for therapeutics and diagnostics. Nat. Rev. Drug Discov. 4(6), 477–488 (2005). Rutishauser U. Polysialic acid at the cell surface: biophysics in service of cell
future science group
interactions and tissue plasticity. J. Cell. Biochem. 70(3), 304–312 (1998). 8
Muhlenhoff M, Eckhardt M, GerardySchahn R. Polysialic acid: three-dimensional structure, biosynthesis and function. Curr. Opin. Struct. Biol. 8(5), 558–564 (1998).
9
Gregoriadis G, Fernandes A, Mccormack B, Mital M, Zhang X. Polysialic acids: potential role in therapeutic constructs. Biotechnol. Genet. Eng. Rev. 16, 203–215 (1999).
10 Gregoriadis G, Fernandes A, Mital M,
Mccormack B. Polysialic acids: potential in improving the stability and pharmacokinetics of proteins and other therapeutics. Cell. Mol. Life Sci. 57(13–14), 1964–1969 (2000). 11 Fernandes AI, Gregoriadis G. The effect of
14 Greco F, Arif I, Botting R et al. Polysialic
acid as a drug carrier: evaluation of a new polysialic acid-epirubicin conjugate and its comparison against established drug carriers. Polym. Chem. 4(5), 1600–1609 (2013). 15 Moghimi SM, Hunter AC, Murray JC.
Long-circulating and target-specific nanoparticles: theory to practice. Pharmacol. Rev. 53(2), 283–318 (2001). 16 Yamauchi H, Yano T, Kato T et al. Effects of
sialic-acid derivative on long circulation time and tumor concentration of liposomes. Int. J. Pharm. 113(2), 141–148 (1995). 17 Bondioli L, Costantino L, Ballestrazzi A
et al. PLGA nanoparticles surface decorated with the sialic acid, N-acetylneuraminic acid. Biomaterials 31(12), 3395–3403 (2010).
polysialylation on the immunogenicity and antigenicity of asparaginase: implication in its pharmacokinetics. Int. J. Pharm. 217(1–2), 215–224 (2001).
18 Bader RA, Silvers AL, Zhang N. Polysialic
12 Emperor design. Product pipeline PolyXen.
19 Wilson DR, Zhang N, Silvers AL, Forstner
Xenetic biosciences (2011). www.xeneticbio.com/polyxen-productpipeline.aspx 13 Valderrama-Rincon JD, Fisher AC, Merritt
JH et al. An engineered eukaryotic protein glycosylation pathway in Escherichia coli. Nat. Chem. Biol. 8(5), 434–436 (2012).
www.future-science.com
acid-based micelles for encapsulation of hydrophobic drugs. Biomacromolecules 12(2), 314–320 (2011). MB, Bader RA. Synthesis and evaluation of cyclosporine A-loaded polysialic acidpolycaprolactone micelles for rheumatoid arthritis. Eur. J. Pharm. Sci. 51, 146–156 (2014). 20 Zhang N, Bader RA: US2012/0294904 A1
(2012).
237
Polysialic acid: overcoming the hurdles of drug delivery “In the coming years, we should expect to see a surge in carrier systems based upon inherently biodegradable, nonimmunogenic, natural materials, such as polysialic acid...” Keywords: drug delivery n nanocarriers n PEG n polysialic acid
“I never really worried about those hurdles. They were just standing there and I was always zooming past them just to get back on the ground again.” – Rodney Milburn (1972 110 m hurdle Olympic champion) In the past two decades, PEG has been lauded as the gold standard material in generating carrier systems that can extend the circularly stability of associated therapeutics and facilitate passive accumulation within diseased tissue [1]. Most investigators take for granted the biocompatibility and aqueous solubility of this synthetic polymer. However, if we step back and take a closer look at PEG, the plausibility that this is the ultimate solution to overcome the hurdles associated with drug delivery is called into question. Although widely regarded as nonimmunogenic, an increasing number of reports suggest that PEG is capable of inducing an immune response in both animal models and human patients, particularly when administered repeatedly. For example, Semple et al. observed a generation of PEG-reactive plasma IgM after dosing mice with PEG-liposomes [2]. Likewise, a recent study by Hamad and coworkers reported concentration- and molecular-weight-dependent activation of the complement system by PEG [3]. When the potential for immunogenicity is combined with a lack of degradability, PEG is a particularly poor choice for drug delivery in the treatment of immune system disorders and/or diseases that require long term treatment, such as rheumatoid arthritis. Of note, low-molecularweight PEG can be metabolized by alcohol and aldehyde dehydrogenase; however, the resultant dicarboxylic acid and hydroxyl-carboxylic acid metabolites are toxic and can lead to acidosis, as indicated in human and animal studies [4]. Natural polymers, with inherent biodegradability and nonimmunogenicity, have been sought after for use in drug delivery as a means
to evade the problems associated with synthetic polymer use, namely, the lack of degradability and the potential of inducing an immune response. Although largely unheralded by other investigators until recent years, in 1993, Gregoriadis posited that polysialic acid (PSA), a highly hydrophilic polysaccharide composed primarily of a-2,8-linked 5-N-glycolyneuraminic acid, could be used as a suitable PEG alternative. Similar to the fixed aqueous layer associated with PEG-modified compounds, PSA is thought to be surrounded by a protective envelope of water, referred to as the ‘watery cloud’, which prevents interactions with cells and proteins [5]. Thus, PSA possesses the requisite properties to impart a stealth nature on associated molecules. In mammalian tissue, neural cell adhesion molecules are post-translationally modified by PSA, and the negatively charged polymer chains are thought to diminish cell–cell interactions, thereby promoting neural plasticity. PSA is assumed to act in a nearly identical fashion to promote tumor growth and cancer metastasis [6]. In bacteria, PSA, with repeating units identical to those found in PSA produced by mammalian cells, is used to mask the invading organism from the host immune system [7,8]. Thus, taken together, PSA acts in nature to reduce undesirable cellular interactions and prevent recognition by the reticuloendothelial system, the exact traits necessary for an effective drug-delivery system. Initia l proof-of-concept studies by Gregoriadis et al. using PSA conjugated to a fluorescent tag verified that the half-life of PSA approached 40 h following intravenous administration to mice [5]. Extending this research to the delivery of therapeutic proteins, the half-lives of asparaginase, insulin, GCSF and IFN-a2b in animal models were significantly increased via polysialylation [9,10]. Moreover, asparaginase did not elicit the immune response that was observed following administration of free asparaginase
10.4155/TDE.13.153 © 2014 Future Science Ltd
Ther. Deliv. (2014) 5(3), 235–237
Rebecca A Bader Author for correspondence: Department of Biomedical & Chemical Engineering, Syracuse Biomaterials Institute, Syracuse University, Syracuse, NY, 13244 USA Tel.: +1 315 857 4622 Fax: +1 315 443 9175 [email protected]
Patricia R Wardwell Department of Biomedical & Chemical Engineering, Syracuse Biomaterials Institute, Syracuse University, Syracuse, NY, 13244, USA
ISSN 2041-5990
235
Editorial | Bader & Wardwell [11]. Based upon polysialylation technology, Gre-
goriadis founded and now serves as Chief Scientific Offer for Xenetic Biosciences (London, UK). Through partnering with biotechnology and pharmaceutical companies, clinical trials are currently being conducted with PSA conjugates of erythropoietin and insulin for the treatment of anemia and diabetes, respectively, while PSA conjugates of factor VIII (hemophilia) and deoxyribonuclease (cystic fibrosis) are in the preclinical stage of development [12]. Although the latter technology relies upon conjugation via standard synthetic methodology, an emerging approach to the synthesis of PSA-modified proteins utilizes the production of recombinant glycoproteins by engineering the protein glycosylation pathways in Escherichia coli [13]. In addition to exploring new methods of synthesizing protein–PSA conjugates, investigators are also looking into how the efficacy of small-molecule therapeutics can be improved via conjugation to PSA [14].
“...polysialic acid acts in nature to reduce undesirable cellular interactions and prevent recognition by the reticuloendothelial system, the exact traits necessary for an effective drug-delivery system.” PSA has also been used in a manner similar to PEG in coating colloidal-carrier systems, which have higher loading capacity and minimal risk of altering drug activity relative to conjugates. Gregoriadis et al. has explored the use of polysialic acid coatings to extend the circulation time of liposomes polysialylation [9,10]. However, some concerns remain pertaining to the utility of PSA coatings. For instance, a change in the conformation of PSA, as can occur following binding to the surface of a carrier system, can lead to an undesirable immunogenic response as a result of a change in the tertiary structure [15]. Notably, coating with sialic acid monomers has largely been unsuccessful in improving the targeted delivery of therapeutics with colloidal carrier systems using animal models, suggesting that PSA, rather than the monomeric form, should be used to extend physiological stability [16,17]. Instead of application of a PSA coating, the authors have focused upon the development of colloidal-carrier systems that utilize PSA as the basis. Micelles have been prepared via self-assembly 236
Ther. Deliv. (2014) 5(3)
following grafting of hydrophobic molecules onto the PSA backbone. Although initial studies used long-chain hydrocarbons, particularly decylamine [18], current research is focused upon use of polycaprolactone (PCL) as the hydrophobic molecule. As expected, based upon Gregoriadis’s ‘watery cloud’ theory, the PSA shell yielded a fixed aqueous layer thickness similar to that obtained for PEG-coated liposomes [19]. The noncytotoxic PSA–PCL system is being explored for use in drug delivery to improve treatment strategies for rheumatoid arthritis and cardiovascular disease. Furthermore, this system can be readily modified by a variety of targeting moieties to facilitate active targeting of diseased tissue. For example, hyaluronic acid has been conjugated to the PSA–PCL surface for enhanced targeting of the hyaluronic acid CD44 receptor, which is overexpressed by certain cell types in cancer, rheumatoid arthritis and cardiovascular disease. PSA has also been used to prepare nanoparticles via complexation with N-trimethylchitosan (TMC). In contrast to existing polysaccharide-based nanoparticles, for the most part, PSA–TMC nanoparticles possess a smaller size, that is more appropriate for drug delivery and a lower polydispersity [20]. The PSA–TMC nanoparticles are being explored for use in the delivery of conventional and new biologic therapeutics for the treatment of rheumatoid arthritis and cystic fibrosis [Wardwell PR, Bader RA, Unpublished Data]. In vivo experiments are currently underway to verify that the PSA-based carrier systems are non-immunogenic and can be used to extend the half-lives and enhance the efficacy of associated therapeutics similar to observations for PSA-conjugates. For a number of years, researchers have seemingly been attempting to overcome the hurdles to drug delivery through the use of a readily available, synthetic polymer, PEG. However, the easiest way to clear a hurdle is not always the best way, particularly for achieving long-term success. Researchers have begun to search nature for the solutions to problems that continue to plague biomaterials, and materials used for drug delivery are not exempt from this search. Although Gregoriadis first suggested PSA as the most rational choice for improved drug delivery 20 years ago, only recently have other investigators truly begun to take notice. In the coming years, we should expect to see a surge in carrier systems based upon inherently biodegradable, nonimmunogenic, natural materials, such as PSA, that can move through the body undetected to better facilitate targeted delivery. future science group
Polysialic acid: overcoming the hurdles of drug delivery Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes
| Editorial
employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
References 1
2
3
4
5
6
7
Fishburn CS. The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics. J. Pharm. Sci. 97(10), 4167–4183 (2008). Semple SC, Harasym TO, Clow KA, Ansell SM, Klimuk SK, Hope MJ. Immunogenicity and rapid blood clearance of liposomes containing polyethylene glycol-lipid conjugates and nucleic acids. J. Pharmacol. Exp. Ther. 312(3), 1020–1026 (2005). Hamad I, Hunter AC, Szebeni J, Moghimi SM. Poly(ethylene glycol)s generate complement activation products in human serum through increased alternative pathway turnover and a MASP-2-dependent process. Mol. Immunol. 46(2), 225–232 (2008). Webster R, Didier E, Harris P et al. PEGylated proteins: evaluation of their safety in the absense of definitive metabolism studies. Drug Metab. Dispos. 35(1), 9–16, (2007). Gregoriadis G, Mccormack B, Wang Z, Lifely R. Polysialic acids: potential in drug delivery. FEBS Lett. 315(3), 271–276 (1993). Dube DH, Bertozzi CR. Glycans in cancer and inflammation. Potential for therapeutics and diagnostics. Nat. Rev. Drug Discov. 4(6), 477–488 (2005). Rutishauser U. Polysialic acid at the cell surface: biophysics in service of cell
future science group
interactions and tissue plasticity. J. Cell. Biochem. 70(3), 304–312 (1998). 8
Muhlenhoff M, Eckhardt M, GerardySchahn R. Polysialic acid: three-dimensional structure, biosynthesis and function. Curr. Opin. Struct. Biol. 8(5), 558–564 (1998).
9
Gregoriadis G, Fernandes A, Mccormack B, Mital M, Zhang X. Polysialic acids: potential role in therapeutic constructs. Biotechnol. Genet. Eng. Rev. 16, 203–215 (1999).
10 Gregoriadis G, Fernandes A, Mital M,
Mccormack B. Polysialic acids: potential in improving the stability and pharmacokinetics of proteins and other therapeutics. Cell. Mol. Life Sci. 57(13–14), 1964–1969 (2000). 11 Fernandes AI, Gregoriadis G. The effect of
14 Greco F, Arif I, Botting R et al. Polysialic
acid as a drug carrier: evaluation of a new polysialic acid-epirubicin conjugate and its comparison against established drug carriers. Polym. Chem. 4(5), 1600–1609 (2013). 15 Moghimi SM, Hunter AC, Murray JC.
Long-circulating and target-specific nanoparticles: theory to practice. Pharmacol. Rev. 53(2), 283–318 (2001). 16 Yamauchi H, Yano T, Kato T et al. Effects of
sialic-acid derivative on long circulation time and tumor concentration of liposomes. Int. J. Pharm. 113(2), 141–148 (1995). 17 Bondioli L, Costantino L, Ballestrazzi A
et al. PLGA nanoparticles surface decorated with the sialic acid, N-acetylneuraminic acid. Biomaterials 31(12), 3395–3403 (2010).
polysialylation on the immunogenicity and antigenicity of asparaginase: implication in its pharmacokinetics. Int. J. Pharm. 217(1–2), 215–224 (2001).
18 Bader RA, Silvers AL, Zhang N. Polysialic
12 Emperor design. Product pipeline PolyXen.
19 Wilson DR, Zhang N, Silvers AL, Forstner
Xenetic biosciences (2011). www.xeneticbio.com/polyxen-productpipeline.aspx 13 Valderrama-Rincon JD, Fisher AC, Merritt
JH et al. An engineered eukaryotic protein glycosylation pathway in Escherichia coli. Nat. Chem. Biol. 8(5), 434–436 (2012).
www.future-science.com
acid-based micelles for encapsulation of hydrophobic drugs. Biomacromolecules 12(2), 314–320 (2011). MB, Bader RA. Synthesis and evaluation of cyclosporine A-loaded polysialic acidpolycaprolactone micelles for rheumatoid arthritis. Eur. J. Pharm. Sci. 51, 146–156 (2014). 20 Zhang N, Bader RA: US2012/0294904 A1
(2012).
237
